The goal of this proposal is to establish in vitro tissue chip models of the closely related neurological disorders tuberous sclerosis complex (TSC) epilepsy, DEPDC5-associated epilepsy, and their associated cardiac dysfunction. The proposed research leverages emerging bioengineering technology for microphysiological systems developed at the Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE) with human induced pluripotent stem cell tools in regular use at Vanderbilt University Medical Center to ask probing questions about genetic disorders that afflict the heart and brain and about the drugs to treat them. The VIIBRE neurovascular unit (NVU)/blood-brain barrier and cardiac I-Wire organ-on-chip models will test the hypothesis that mTORC1 and mTORC2 signaling differentially affect neural and cardiac dysfunction in TSC- and DEPDC5-associated epilepsy. The primary and shared abnormality in patients with TSC and DEPDC5- associated epilepsy is dysregulation of the mTOR kinase complex 1 (mTORC1) signaling pathway. TSC also has abnormalities in mTORC2 signaling not seen in DEPDC5-associated epilepsy. A focus on mTOR signaling in these human mTORopathies has several advantages. First, rapamycin and related compounds are FDA- approved mTORC1 inhibitors and have been shown to have efficacy in some aspects of the disease manifestations of TSC. Second, TSC- and DEPDC5-associated epilepsy are both associated with neural and cardiac dysfunction. Third, the role for compensatory or differential mTORC2 activity is unclear and controversial. For patients with TSC, drugs targeting the mTORC1 signaling pathway have been associated with shrinkage of brain tumors, reduced seizures, and improved cardiac function. Thus, drug development for this group of diseases is well suited for study using both the NVU and I-Wire cardiac-tissue chips. In its first two years, the project will develop the NVU and I-Wire disease models, aimed at refining the TSC and DEPDC5 NVU model; applying the I-Wire model to TSC and DEPDC5 cardiomyocytes; and validating outcome methodologies in control and patient-derived NVU and I-Wire chips. The next three years aim to evaluate, for biomarker identification in control, TSC, and DEPDC5 NVU and I-Wire chips, changes in mTORC1 and mTORC2 signaling, protein markers of cellular health and toxicity, metabolites, functional measures and electrophysiological activity; and, use ion mobility-mass spectrometry to evaluate NVU and I-Wire outcome measures plus drug metabolites after treatment with mTORC1 inhibitor rapamycin, the seizure drug vigabatrin, and novel pre-clinical mTOR drug candidates. The NVU and I-Wire will assess the efficacy and toxicity of these agents and define TSC/DEPDC5 shared vs disease-specific effects. With this organ-on-chip/human induced pluripotent stem cell platform, it will be possible to address currently confounding mechanisms of pathogenesis, identify new disease biomarkers, quantify how drugs cross the normal and diseased blood-brain barrier, and ultimately develop effective therapies and hence enable bench-to-bedside translation.